Signals discharged from the B-cell antigen receptor (BCR) control B-cell physiological responses by regulating specific transcription factors and their downstream target genes. Our long-term goal is to understand the intracellular signal transduction pathways that regulate genes under BCR control. Transcription factor NF-kappaB is coupled to the BCR and essential for B cell survival and proliferation. However, the mechanisms that regulate the BCR/NF-KB axis remain poorly understood. The applicant's laboratory has recently discovered that Bruton's tyrosine kinase (BTK), phospholipase C-gamma2 (PLC-gamma2), and IkappaB kinase (IKK) are all integral components of this pathway. Importantly, interference with NF-kappaB signaling at the level of BTK leads to B cell immunodeficiencies in mice (X-linked immunodeficiency; xid) and humans (X-linked agammaglobulinemia; XLA). This grant application is predicated on the central hypothesis that BTK stimulates PLC-gamma2 phosphorylation and activation, which leads to calcium mobilization and protein kinase C (PKC) induction. In turn, these second messengers act in concert to stimulate the effectors that activate BCRresponsive IRK complexes. To test the central hypothesis, we propose an integrated series of biochemical studies with mutant B cells that lack either BTK, PLC-gamma2, or IKK. Studies described in Specific Aim 1 will elucidate the biochemical interplay between BTK and PLC-gamma2 as well as the PKC isoforms that affect IKK function in response to BCR engagement. Specific Aim 2 experiments will define the molecular size, phosphorylation status, and subunit composition of IKK complexes under BCR control. In Specific Aim 3, studies are proposed to identify the precise BCR-inducible modifications to IKK that lead to NF-kappaB activation. Results from the proposed studies will uncover novel regulatory steps in the BCR/NF-kappaB axis that are crucial for the development of an adaptive immune response. Identification of these important missing links may reveal new immunotherapeutic targets for intervention in B-cell deficiency diseases such as XLA.